In the vaccine art antigens are introduced into an organism in a manner so as to stimulate an immune response in the host organism. The induction of an immune response depends on many factors among which are believed to be the chemical composition and configuration of the antigen, the potential of the immune system of the challenged organism, and the manner and period of administration of the antigen. An immune response has many aspects some of which are exhibited by the cells of the immune system, (e.g., B-lymphocytes, T-lymphocytes, macrophages, and plasma cells). Immune system cells may participate in the immune response through interaction with antigen, interaction with other cells of the immune system, the release of cytokines and reactivity to those cytokines. Immune response is conveniently (but arbitrarily) divided into two main categories--humoral and cell-mediated. The humoral component of the immune response includes production of immunoglobulins specific for the antigen. The cell-mediated component includes the generation of delayed-type hypersensitivity and cytotoxic effector cells against the antigen.
In some instances immune response is the result of an initial or priming dose of an antigen that is followed by one or more booster exposures to the antigen. Priming with relatively strong immunogens and liposomes is discussed in "Liposomal Enhancement of the Immunogenicity of Adenovirus Type 5 Hexon and Fiber Vaccines", Kramp, W. J. et al., Infection and Immunity, 25:771-773 (1979) and "Liposomes as Adjuvants with Immunopurified Tetanus Toxoid: the Immune Response", Davis, D. et al., Immunology Letters, 14:341-8 (1986/1987).
Ideally, an antigen will exhibit two properties, the capacity to stimulate the formation of the corresponding antibodies and the propensity to react specifically with these antibodies. Antigens bear one or more epitopes which are the smallest part of an antigen recognizable by the combining site of an antibody.
In particular instances antigens or fractions antigens with particular presenting conditions, the immune response precipitated by the desired antigen is inadequate or nonexistent and insufficient immunity is produced. This is particularly the case with peptides or other small molecules used as antigens.
In such cases the vaccine art recognizes the use of substances called adjuvants to potentiate an immune response when used in conjunction with an antigen or immunogen. Adjuvants are further used to an elicit immune response sooner, or a greater response, or with less antigen or immunogen or to increase production of certain antibody subclasses that afford immunological protection, or to enhance components of the immune response (e.g., humoral, cellular). Liposomal vaccines and adjuvancy are further discussed in U.S. patent application Ser. No. 07/397,777, now abandoned, to Popescu et al., filed on date even herewith the teachings of which are incorporated herein by reference.
Known adjuvants are Freund's Adjuvants (and other oil emulsions Bortedella Pertussis, Lipid A (the glycophospholipid moiety of lipopolysaccharide found in Gram-negative bacteria), aluminum salts (and other metal salts), Mycobacterial products (including muramyl dipeptides), and liposomes. As used herein the term "adjuvant" will be understood to mean a substance or material administered together or in conjunction with an antigen which increases the immune response to that antigen. Adjuvants may be in a number of forms including emulsion (e.g., Freund's adjuvant) gels (aluminum hydroxide gel) and particles (liposomes) or as a solid material.
It is believed that adjuvant activity can be affected by a number of factors. Among such factors are (a) carrier effect, (b) depot formation, (c) altered lymphocyte recirculation, (d) stimulation of T-lymphocytes, (e) direct stimulation of B-lymphocytes and (f) stimulation of macrophages.
With many adjuvants adverse reactions are seen. In some instances adverse reactions include granuloma formation at the site of injection, severe inflammation at the site of injection, pyrogenicity, adjuvant induced arthritis or other autoimmune response, or oncogenic response. Such reactions have hampered the use of adjuvants such as Freund's adjuvant.
In particular embodiments liposome adjuvants are utilized. U.S. Pat. No. 4,053,585 issued Oct. 17, 1977 to Allison et al. states that liposomes of a particular charge are adjuvants.
Other substances such as immunomodulators (e.g., cytokines such as the interleukins) may be combined in adjuvants as well.
Humoral immune response may be measured by many well known methods. Single Radial Immunodifussion Assay (SRID), Enzyme Immunoassay (EIA) and Hemagglutination Inhibition Assay (HAI) are but a few of the commonly used assays.
EIA, also known as ELISA (Enzyme Linked Immunoassay), is used to determine total antibodies in a sample. The antigen is adsorbed to the surface of a microtiter plate. The test serum is exposed to the plate followed by an enzyme linked immunogloublin, such as IgG. The enzyme activity adherent to the plate is quantified by any convenient means such as spectrophotometry and is proportional to the concentration of antibody directed against the antigen present in the test sample.
Tests to measure cellular immune response include determination of delayed-type hypersensitivity or measuring the proliferative response of lymphocytes to target antigen.
Liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. Liposomes may be unilamellar vesicles (possessing a single bilayer membrane) or multilameller vesicles (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer). The bilayer is composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "heads" region. The structure of the membrane bilayer is such that the hydrophobic (nonpolar) "tails" of the lipid monolayers orient toward the center of the bilayer while the hydrophilic "head" orient towards the aqueous phase.
The original liposome preparation of Bangham, et al. (J. Mol. Biol., 1965, 13:238-252) involves suspending phospholipids in an organic solvent which is then evaporated to dryness leaving a phospholipid film on the reaction vessel. Next, an appropriate amount of aqueous phase is added, the mixture is allowed to "swell," and the resulting liposomes which consist of multilamellar vesicles (MLVs) are dispersed by mechanical means. This technique provides the basis for the development of the small sonicated unilamellar vesicles described by Papahadjopoulos et al. (Biochim. Biophys. Acta., 1968, 135:624-638), and large unilamellar vesicles.
Unilamellar vesicles may be produced using an extrusion apparatus by a method described in Cullis et al., PCT Application No. WO 86/00238, published Jan. 16, 1986, entitled "Extrusion Technique for Producing Unilamellar Vesicles" incorporated herein by reference. Vesicles made by this technique, called LUVETS, are extruded under pressure once or a number of times through a membrane filter. LUVETs will be understood to be included in the term "unilamellar vesicle".
Another class of liposomes are those characterized as having substantially equal lamellar solute distribution. This class of liposomes is denominated as stable plurilamellar vesicles (SPLV) as defined in U.S. Pat. No. 4,522,803 to Lenk, et al., monophasic vesicles (MPVs) as described in U.S. Pat. No. 4,588,578 to Fountain, et al. and frozen and thawed multilamellar vesicles (FATMLV) wherein the vesicles are exposed to at least one freeze and thaw cycle, as described in Bally et al., PCT Publication No. 87/00043, Jan. 15, 1987, entitled "Multilamellar Liposomes Having Improved Trapping Efficiencies" corresponding to U.S. Pat. No. 4,975,282. U.S. Pat. No. 4,721,612 to Janoff et al. describes steroidal liposomes for a variety of uses. The teachings of these references as to the preparation and use of liposomes are incorporated herein by reference.
Lipids of net negative charge are well known in the art and include for example, phosphatidyserine, phosphatidic acid, and phosphatidylglycerol. Lipids of net positive charge are well known in the art and include for example, aminodiglycerides, glyceridecholine, stearylamine, trimethylstearylamine, and dioctadecyl trimethylamonnio propane. In general any bilayer forming amphiphile which has a charged hydrophilic moiety may be used.
In addition, lipid charge may be manipulated by a number of methods well known in the art, such as by linking the lipid to a moiety of appropriate net charge. For example, the neutral lipid cholesterol may be linked to succinic acid (negative charge) to yield cholesterol hemisuccinate (CHS) of negative charge. The tris(hydroxymethyl)aminomethane form of CHS is designated CHS.sub.tris its application to liposomes is more fully discussed in U.S. Pat. No. 4,721,612 the teachings of which are incorporated herein by reference.